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University Mk:rorilms International 300 N. Zeeb Road Ann Arbor, Ml 48106 8300200 Alves de Lima, Marinez Muraro PHYSICAL AND CHEMICAL FACTORS AFFECTING REPRODUCTION OF PRATYLENCHUS SPECIES IN MONOXENIC CULTURE The Ohio State University Ph.D. 1982 University Microfilms International300 N. Zeeb Road, Ann Arbor, MI 48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a checkV . mark 1. Glossy photographs or pages^ 2. Colored illustrations, paper or print^ 3. Photographs with dark background_____ 4. Illustrations are poor co_______ p y 5. Pages with btack marks, not original _copy 6. Print shows through as there is text on both sides_______ of page 7. Indistinct, broken or small print on several______ pages 8. Print exceeds margin requirements______ 9. Tightly bound copy with print lost______ in spine 10. Computer printout pages with indistinct______ print 11. P ag e(s)_____________lacking when material received, and not available from school or author. 12. P ag e(s)_____________seem to be missing in numbering only as text follows. 13. Two pages numbered_____________. Text follows. 14. Curling and wrinkled p______ a g e s 15. O ther____________________________________________________________________________ University Microfilms International PHYSICAL AND CHEMICAL FACTORS AFFECTING REPRODUCTION OF PRATYLENCHUS SPECIES IN MONOXENIC CULTURE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University by Marinez Muraro Alves de Lima, B.S., M.S. ***** The Ohio State University 1982 Reading Committee: Approved By Dr. R. M. Riedel Dr. L. Rhodes Advisor Dr. I, Deep Department of Plant Pathology This dissertation is dedicated to my parents, Isaltina and Roberto, and to my husband, Abdalla, for their constant concern, encouragement and support. ACKNOWLEDGEMENTS I thank my colleagues, the graduate students, staff and faculty members of the Department of Plant Pathology for providing such a warm and friendly environment conducive to learning. I extend my sincere thanks to Dr. R. M. Riedel for his advice and criticism during the conduction of the research and review of the manuscript; to Dr. M. J. Martin for her suggestions, supply of nematode stock cultures and incentive during the research; and to R. D. Thames for her generous help and encouragement. My sincere appreciation is reserved to the Instituto Agronomico de Campinas, Bnpresa Brasileira de Pesquisa Agropecuaria - EMBRAPA and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico - CNPq, for granting me this invaluable opportunity to further my education. VITA November 16, 1944 Born - Sao Paulo-SP, Brazil 1967 B.S., Escola Superior de Agricultura "Luiz de Queiroz," Piracicaba-SP, Brazil 1980 M.S. The Ohio State University, Columbus, Ohio FIELD OF STUDY Major Field: Plant Pathology Research Interest: Plant Tissue Culture Applied to Plant Pathology TABLE OF CONTENTS Page DEDICATION........................................... ii ACKNOWLEDGEMENTS...................................... iii VITA ................................................ iv LIST OF.......... TABLES ............................. vi LIST OF FIGURES............................... viii LIST OF PLATES............................. ix INTRODUCTION ......................................... 1 Chapter I. PHYSICAL AND CHEMICAL FACTORS AFFECTING REPRODUCTION OF PRATYLENCHUS PENETRANS IN MONOXENIC CULTURE........ 3 Introduction and Literature Review............... 3 Materials and Methods......................... 9 Results.................................... 16 Discussion.................................. 37 II. COMPARATIVE REPRODUCTION OF FOUR SPECIES OF PRATYLENCHUS ON ALFALFA CULTURE........................ 47 Introduction and Literature Review............... 47 Materials and Methods ......................... 49 Results.................................... 52 Discussion................................... 60 BIBLIOGRAPHY......................................... 65 APPENDIX -- Composition of Riedel's et al. (1973) and Murashige and Skoog (196‘ZJ’ me d i a ............... 71 v LIST OF TABLES Reproduction of Pratylenchus penetrans on four plant tissues cultured on three different media............. 20 Reproduction of Pratylenchus penetrans on alfalfa seedlings cultured on Riedel's et alY medium at three yeast extract concentrations ....................... 21 Reproduction of Pratylenchus penetrans on alfalfa seedlings cultured on Riedelfs et al. medium at three yeast extract concentrations........................ 22 Reproduction and composition of Pratylenchus penetrans on alfalfa seedlings cultured on Riedel's et al. medium at three yeast extract concentrations................. 23 Reproduction of Pratylenchus penetrans on pea roots cv. 'Extra Early Alaska' cultured on Riedel's et al. medium at three yeast extract concentrations........... 24 Reproduction of Pratylenchus penetrans on pea roots on pea roots of two cultivars cultured on Riedel’s et aL. medium..................................... 25 Populations of Pratylenchus penetrans on alfalfa seedlings cultured on Riedel's et al. medium in three different kinds of containers.................. 26 Populations of Pratylenchus penetrans on alfalfa seedlings cultured on Riedel*s et al. medium in different kinds of containers....................... 27 Population density of Pratylenchus penetrans on alfalfa seedlings cultured on Riedel's et al. medium in different kinds of containers .................... 28 Reproduction of four Pratylenchus species on alfalfa callus cultured on Riedel's et al. medium at 25 C for 100 days .................................... 54 Reproduction of four Pratylenchus species on alfalfa on alfalfa callus cultured on Riedel’s et al. medium at 25 C for 120 days............................... 55 vi Table Page 3.1. Composition of Riedel*s et al. medium (1973)......... 72 3.2. Composition of Murashige and Skoog's medium (1962)............................................ 73 vii LIST OF FIGURES Figure Page 1.1. Reproduction of Pratylenchus penetrans on Riedel's et al. medium with 1 g yeast extract/1 at 25 C. A) for a 80-day period; B) for a 110-day period.......... 29 1.2. Reproduction of Pratylenchus penetrans on Riedel's et al. medium with 0.5 g yeast extract/1 at 25 C. Aj “For a 80-day period; B) for a 110-day p e r i o d......... 30 1.3. Reproduction of Pratylenchus penetrans on Riedel's et al. medium without yeastextract at 25 C . A7 Tor a 80-day period; B) for a 110-day period.......... 31 2.1. Reproduction of Pratylenchus brachyurus on Riedel's et al. medium at“25 C. A) Tor a 100-day period; For a 120-day p e r i o d ............................. 56 2.2. Reproduction of Pratylenchus crenatus on Riedel's et al. medium at 25 C. A) for a 100-day period; B] Tor a 120-day p e r i o d ............................. 57 2.3. Reproduction of Pratylenchus penetrans on Riedel's et al. medium at 25 C. A] for a- lOO’-day period; B) for a 120-day period ............................. 58 2.4. Reproduction of Pratylenchus scribneri on Riedel's et al. medium at'""25 C. A) for a 100-day period; B) Tor a 12-day pe r i o d ............................... 59 viii LIST OF PLATES Plate Page I. Inoculation of aseptic nematode suspension into French square bottles in a laminar airflow cabinet........................................... 13 II. Baermann funnels made from styrofoam cups and cheese-cloth used for nematode extraction............